GSM8002512: PSC27-RAD-2; Homo sapiens; OTHER
Starting material cells were harvested under 3 different biological conditions: proliferating (CTRL), radiation-treated (RAD) and chemotherapeutically damaged (BLEO). CUT&Tag assay was performed as previously described. Briefly, 500,000 cells were washed and incubated with concanavalin A coated magnetic beads. Cell-magnetic beads complex were resuspended in Dig-wash Buffer, and then the primary antibody of CTCF was diluted 1:50 in Dig-Wash buffer and then incubated on a rotator overnight at 4 °C. The next day, cells were incubated with an appropriate secondary antibody (goat anti-rabbit) (1:100) in Dig-Wash buffer. Then, cells were incubated with pAG-Tn5 (1:200) in Dig-300 Buffer. For tagmentation, bead bound cells were resuspended in 50 μl of tagmentation buffer, with 1 μl of 10% SDS then added directly into the reaction to end the tagmentation. DNA was purified by 1.5 × Ampure XP beads. PCR cycling conditions: 72 °C for 5 min; 98 °C for 30 s; 12 cycles of 98 °C for 10 s and 63 °C for 30 s; final extension at 72 °C for 1 min. Libraries were sequenced (150-bp paired ends) by on the Illumina NovaSeq 6000. Genomic DNA libraries were prepared for sequencing using standard Illumina protocols. One library was generated from each individual sample per treatment group.